Photodynamic therapy (PDT) is a treatment technique that uses a photosensitizing dye which localizes at, or near, the treatment site, and when irradiated in the presence of oxygen serves to produce cytotoxic materials, such as singlet oxygen (O.sub.2 (.sup.1 .DELTA..sub.g)), from benign precursors (e.g. (O.sub.2 (.sup.3 .SIGMA..sub.g -)). Other reactive species such as superoxide, hydroperoxyl, or hydroxyl radicals may be involved. At the doses used, neither the light nor the drug has any independent biological effect. In PDT, the photosensitizer acts in a `catalytic` way, since its function is not to react directly with the cellular targets, but to absorb light energy and to transfer it to molecular oxygen, regenerating ground state photosensitizer.
The effectiveness of PDT is due to three additional factors: i) The photosensitive dyes used in PDT must have the ability to localize at the treatment site relative to surrounding tissue. ii) The high reactivity and short lifetime of activated oxygen means that it has a very short range and is unlikely to escape from the cell in which it is produced; cytotoxicity is therefore restricted to the precise region of tissue absorbing light, perhaps down to the cellular level. iii) Developments in lasers and fiber optics allow a beam of intense light to be delivered precisely to many parts of the body.
For reviews of photodynamic therapy, see U.S. Pat. No. 5,252,720 (incorporated by reference herein); Sindelar et al., (1991); Grossweiner, L. I., (1991); Henderson, B. W. and T. J. Dougherty, (1992); and Moan, J. and K. Berg, (1992). In recent years, considerable effort has been devoted to the synthesis and study of new photosensitizers (a review is found in Brown, S. B. and Truscott, T. G., 1993). The development of more effective photochemotherapeutic agents requires the synthesis of compounds which absorb in the spectral region where living tissues are relatively transparent (i.e., 700-1000 nm), have high triplet quantum yields, and are minimally toxic.
An effective photo-catalyst for PDT and DNA cleavage would have the following properties:
1. Easily available PA1 2. Low intrinsic toxicity PA1 3. Long wavelength absorption PA1 4. Efficient photosensitizer for singlet oxygen production PA1 5. Fair solubility in water PA1 6. Selective uptake in lipophilic tissue such as atheroma or tumor tissue PA1 7. Showing high affinity for enveloped viruses PA1 8. Quick degradation and/or elimination after use PA1 9. Chemically pure and stable PA1 10. Easily subject to synthetic modification PA1 11. Efficient at physiological temperature and pH PA1 12. Specific for certain biological substrates PA1 13. Easy administered to a biological system
Photodynamic cleavage of DNA is known. For example, Praseuth et al., reported cleavage of plasmid DNA by synthetic water-soluble porphyrins with visible light in the presence of oxygen. Fiel, R. J. (1989) also reported the photosensitized strand cleavage and oxidative-reductive strand scission of DNA by iron porphyrins. In another example, Kobayashi et al. reported cleavage of plasmid DNA by sodium pheophorbide (a derivative of chlorophyll) with visible light in the presence of oxygen. Porphyrin-oligonucleotide derivatives were reportedly used to effect sequence specific modifications of DNA substrates followed by cleavage using hot piperidine (Vlassov et al., 1991; Le Doan et al., 1990). The absorption wavelengths for these porphyrin conjugates were below 700 nm, a range that does not penetrate tissue as effectively as longer wavelengths of light.
The use of ultraviolet light with the drug 8-methoxy-psoralen to treat psoriasis is well established. Lee et al. relates to the interaction of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with single-stranded DNA. Crosslinked photoadducts between pyrimidines and psoralen appear to form. This treatment may result in the development of cancerous cells. Furthermore, irradiation at the short wavelength of about 365 nm does not penetrate the body and is therefore only useful on the body surface. Psoralen-based treatments must allow the drug to leave the body before the patient is exposed to visible light or the reaction will continue on the skin surface.
Sequence-specific cleavage of DNA has also been reported for dark reactions using oligonucleotides derivatized with metal complexes. Some examples include oligonucleotide-EDTA-Fe complexes (Strobel, D. A. and P. B. Dervan, 1989; Lin, et al., 1989; Dreyer, G. B. and P. B. Dervan, 1985), oligonucleotide-tricationic porphyrins with metal binding appendages (Groves, J. T. and T. P. Farrell, 1989), oligonucleotide-phenanthroline-copper complexes (Chen, C. H. B. and D. S. Sigman, 1988), oligonucleotide-manganese-porphyrins (Meunier, B. et al., 1993), and iron-porphyrins linked to oligonucleotides (Le Doan et al., 1986, 1987).
All of the above examples of DNA cleavage have been directed to cleavage of single-stranded DNA. However, since cellular DNA is predominantly double-stranded, for practical use and effect in most instances it would be desirable to cleave double-stranded DNA. Oligonucleotide-derived therapeutics that target double-stranded DNA are referred to as anti-gene agents. Binding to double-stranded DNA is much less straight-forward than binding to RNA or single-stranded DNA (the antisense approach), since special Hoogsteen interactions must be used. Once bound, the anti-gene agent must be able to compete successfully with cellular factors in order to inhibit gene expression.
______________________________________ LIST OF ABBREVIATIONS ______________________________________ DCA Dichloroacetic acid DCC Dicyclohexylcarbodiimide DMAP Dimethylaminopyridine DMF Dimethylformamide DMT Dimethoxytrityl protecting group DMT-Cl Dimethoxytrityl chloride EDC L-Ethyl-3-[3-(dimethylamino)propyl] carbodiimide EDTA Ethylenediamine tetraacetic acid IPA Isopropylalcohol NHS N-hydroxysuccinimide NM Nanometers PTSA p-Toluenesulfonic acid monohydrate TEA Triethylamine TEAB Triethylammonium bicarbonate TFA Trifluoroacetic acid TsCl Tosyl chloride THF Tetrahydrofuran TXP(Tx) Texaphyrin ______________________________________